CN114042160B - Application of CTD-2256P15.2 and its encoded micropeptides as targets in the development of tumor therapeutic drugs - Google Patents

Abstract

The invention discloses the application of CTD‑2256P15.2 and its coded micropeptide as a target in the development of tumor treatment drugs. The invention provides the following uses of CTD-2256P15.2 inhibitors: 1) preparing tumor treatment products; 2) preparing products that reduce the drug resistance of tumor cells to chemotherapeutic drugs; 3) preparing and improving the sensitivity of tumor cells to chemotherapeutic drugs products; CTD-2256P15.2 or the inhibitor of the encoded micropeptide PACMP provided by the present invention can significantly inhibit the growth of tumor cells, increase tumor cell apoptosis, and reduce tumor volume when acting on tumor cells or tumor tissues , has excellent antitumor effect.

Description

Application of CTD-2256P15.2 and its encoded micropeptide as targets in the development of tumor therapeutic drugs

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to application of CTD-2256P15.2 and its encoded micropeptide as targets in the development of tumor therapeutic drugs.

Background Art

DNA damaging agents are often used in cancer treatment, such as radiotherapy and many chemotherapy drugs, which mainly induce DNA damage in cancer cells, inhibit cancer cell proliferation, and induce their death. However, tumor cells can often resist the effects of radiotherapy and chemotherapy drugs by changing their own DNA damage repair function, resulting in tumor resistance (including primary and secondary resistance). PARP inhibitors (PARPi) are the first drugs that have successfully used the concept of synthetic lethality to be approved for clinical use, and have achieved good therapeutic effects in the treatment of tumor patients with BRCA1/2 mutations. It is currently believed that PARPi can inhibit the catalytic activity and single-strand break repair of poly ADP polymerases such as PARP1, leading to the accumulation of DNA double-strand breaks (DSBs), or capture PARP1/2 on the DNA chain, interfering with its timely removal from the damage site, thereby inducing replication stress and DSB damage. After PARPi is given to tumor patients carrying BRCA1 or BRCA2 mutations, DSBs cannot be effectively repaired due to the inhibition of the DNA homologous recombination repair pathway, leading to cell death. However, similar to chemotherapy drugs widely used in clinical practice, the use of PARPi also leads to drug resistance, which is largely caused by the recovery of DNA damage repair function. The development of these drug resistances seriously restricts the clinical use and efficacy of PARPi. It is currently known that more than 40% of ovarian cancer patients with BRCA mutations cannot benefit from PAPRi treatment. Therefore, finding key molecules that regulate DNA damage response, chemotherapy drugs and PARPi resistance will not only help discover prognostic biomarkers, but also help discover new targets, develop combination therapies, and improve the efficacy of tumor treatment.

Long noncoding RNA (lncRNA) is a type of noncoding RNA with a length greater than 200 nt in cells. It plays an important regulatory role in a variety of physiological and pathological processes, and its abnormal expression is often closely related to tumor occurrence, development and prognosis. Studies have found that some lncRNAs can regulate the DNA damage repair process, such as NORAD, DDSR1 and lnc BGL3. However, these studies have focused on lncRNA as a molecular scaffold to bind specific proteins to regulate DNA repair. Compared with the diverse mechanisms of action and important regulatory functions of lncRNA, little is known about lncRNA related to DNA damage repair and tumor chemotherapy sensitivity. In recent years, more and more research evidence has shown that lncRNA can encode micropeptides with biological activity, thereby regulating tumor growth, invasion and metastasis. However, to date, there has been no report on whether micropeptides encoded by lncRNA regulate DNA damage repair and tumor chemotherapy resistance.

Summary of the invention

One object of the present invention is to provide uses of CTD-2256P15.2 inhibitors.

The present invention provides an inhibitor having any of the following functions a-d in at least one of the following 1)-9):

1) Preparation of tumor treatment products;

2) Preparation of products that reduce the resistance of tumor cells to chemotherapy drugs;

3) Preparation of products that increase the sensitivity of tumor cells to chemotherapeutic drugs;

4) Preparation of products that inhibit the DNA homologous recombination repair pathway in tumor cells;

5) Preparation of products that inhibit the microhomology-mediated end joining repair pathway in tumor cells;

6) Preparation of products for inhibiting DNA damage-induced poly ADP-ribose chain formation;

7) Preparation of products that inhibit tumor drug resistance caused by the DNA homologous recombination repair pathway;

8) Preparation of products that inhibit tumor drug resistance induced by the microhomology-mediated end-joining repair pathway in tumor cells;

9) Preparation of products that inhibit tumor drug resistance caused by DNA damage-induced poly ADP-ribose chain generation;

a. Inhibit the expression of CTD-2256P15.2 gene;

b. Inhibit the biological function of the micropeptide PACMP encoded by the CTD-2256P15.2 gene;

c. Inhibit the biological function of fusion proteins containing PACMP;

d. Inhibit the biological function of the complex containing PACMP;

The nucleotide sequence of the CTD-2256P15.2 gene is sequence 1 or positions 138-272 of sequence 1 in the sequence list.

The amino acid sequence of the micropeptide PACMP is sequence 8 in the sequence list.

The above-mentioned tumor treatment products are tumor treatment drugs, and their functions include inhibiting the proliferation of tumor cells or inducing tumor cell death or inhibiting tumor growth.

Furthermore, in the tumor treatment drug, CTD-2256P15.2 or an inhibitor of the micropeptide PACMP encoded by it is used as the only active ingredient or one of the active ingredients.

In the above applications, the tumors include but are not limited to breast cancer, ovarian cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, head and neck cancer, bladder cancer, cervical cancer, diffuse B large cell lymphoma, esophageal cancer, glioma, pancreatic cancer, prostate cancer, melanoma, thymoma, and endometrial cancer.

In the above application, the inhibitor refers to a molecule that can inhibit CTD-2256P15.2 or its encoded micropeptide PACMP, and the inhibitory effect includes but is not limited to: inhibiting the transcription or translation of CTD-2256P15.2, promoting the degradation of CTD-2256P15.2 or PACMP, and inhibiting the function of PACMP.

The inhibitor of CTD-2256P15.2 or micropeptide PACMP can be siRNA, shRNA, antisense RNA, miRNA, gene knockout or knockdown CRISPR-related plasmid and viral vector, antibody, peptide, small molecule compound.

Specifically, the inhibitor is a siRNA or shRNA targeting CTD-2256P15.2 or a gRNA targeting the PACMP coding region or a vector expressing the above respective RNA;

The nucleotide sequence of the siRNA targeting CTD-2256P15.2 RNA is sequence 2 or sequence 3 in the sequence list;

The nucleotide sequence of the shRNA targeting CTD-2256P15.2 RNA is sequence 4 or sequence 5 in the sequence list;

The nucleotide sequence of the gRNA targeting the PACMP coding region is sequence 6.

The function of the siRNA and shRNA for inhibiting CTD-2256P15.2 provided by the present invention refers to the RNA sequence targeting CTD-2256P15.2 and reducing its RNA level, including the above sequence and all siRNA and shRNA with similar functions.

The use of the above inhibitors and other anti-tumor substances (i.e., novel anti-tumor drug combination solutions) in at least one of the following:

1) Preparation of tumor treatment products;

2) Preparation of products that reduce the resistance of tumor cells to chemotherapy drugs;

3) Preparation of products that increase the sensitivity of tumor cells to chemotherapeutic drugs;

The other anti-tumor substances are reagents or instruments required for other anti-tumor drugs or other anti-tumor treatment methods. The other tumor treatment drugs or methods refer to tumor treatment drugs or methods that can damage tumor cell DNA or induce replication stress, including but not limited to: anthracyclines, camptothecin, PARP inhibitors, ATR inhibitors, CDK4/6 inhibitors, and radiotherapy.

Furthermore, the anti-tumor drug is a chemotherapy drug.

The novel anti-tumor drug combination regimen can be in any of the following forms:

(1) CTD-2256P15.2 or an inhibitor of the micropeptide PACMP encoded by it is administered separately from other tumor therapeutic drugs or treatment methods. The administration routes can be the same or different. The above inhibitor can be administered independently before, during or after the administration of other tumor therapeutic drugs or treatment methods.

(2) CTD-2256P15.2 or the inhibitor of the micropeptide PACMP encoded by it is prepared into a compound preparation with other tumor therapeutic drugs, that is, when CTD-2256P15.2 or the inhibitor of the micropeptide PACMP encoded by it and other tumor therapeutic drugs are administered simultaneously by the same administration route, the two are prepared into a compound preparation.

Another object of the present invention is to provide a product, which includes the above inhibitor, and other tumor therapeutic drugs or reagents or instruments required for other anti-tumor therapeutic methods;

The product has at least one of the following functions:

1) Cancer treatment;

2) Reduce the resistance of tumor cells to chemotherapy drugs;

3) Increase the sensitivity of tumor cells to chemotherapy drugs.

The use of the above-mentioned CTD-2256P15.2 or the micropeptide PACMP encoded therein as a target for treating tumors is also within the scope of protection of the present invention.

The use of the above-mentioned CTD-2256P15.2 gene as a marker in the preparation of a product for evaluating the sensitivity of tumor patients to chemotherapy drugs or predicting the prognosis of tumor patients is within the scope of protection of the present invention.

Another object of the present invention is to provide a use of a substance for detecting the expression level of CTD-2256P15.2 in tumor tissue.

The application of the substance for detecting the expression amount of CTD-2256P15.2 in tumor tissue provided by the present invention is as follows:

1) Prepare products to predict the prognosis of tumor patients after chemotherapy;

2) Prepare products for evaluating or assisting in evaluating the sensitivity of cancer patients to chemotherapy drugs.

The present invention also provides an application of a substance and a data processing device for detecting the expression amount of CTD-2256P15.2 in tumor tissue, which is any one of 1)-2):

1) Prepare products to predict the prognosis of tumor patients after chemotherapy;

2) Prepare products for evaluating or assisting in evaluating the sensitivity of tumor patients to chemotherapy drugs;

The data processing device has a built-in module; the module has the following functions as shown in (a1) and (a2):

(a1) using ex vivo tumor tissues of a test group consisting of tumor patients as specimens, determining the expression level of the CTD-2256P15.2 gene in each specimen, and then dividing the test group into a low expression group and a high expression group according to the gene expression level;

(a2) determining the prognosis of the patient to be tested from the test population according to the following criteria:

The prognosis of the patients to be tested in the low expression group after chemotherapy is better or potentially better than that of the patients to be tested in the high expression group;

Or, the prognostic overall survival of the patients to be tested in the low expression group after chemotherapy is longer or potentially longer than that of the patients to be tested in the high expression group;

Or, the overall survival rate of the patients to be tested in the low expression group after chemotherapy is higher or potentially higher than that of the patients to be tested in the high expression group;

Or, the prognosis of the patients to be tested in the low expression group after chemotherapy is longer or potentially longer than that of the patients to be tested in the high expression group;

Or, the prognosis of the patients to be tested in the low expression group after chemotherapy is higher or potentially higher than that of the patients to be tested in the high expression group;

Or, the sensitivity of the patients to be tested in the low expression group to chemotherapy drugs is higher or may be higher than that of the patients to be tested in the high expression group.

The present invention also provides a system for predicting the prognosis of a tumor patient to be tested after chemotherapy, comprising a substance for detecting the expression amount of CTD-2256P15.2 in tumor tissue and the above-mentioned data processing device.

The substance for detecting the expression level of CTD-2256P15.2 in tumor tissue is a probe or primer that specifically binds to or amplifies the CTD-2256P15.2.

The chemotherapy drugs used in the above chemotherapy are tumor treatment drugs that can cause DNA damage, including but not limited to anthracyclines, camptothecin or PARP inhibitors.

In the reagent for evaluating the sensitivity of tumor patients to chemotherapy drugs or the prognosis, the expression of CTD-2256P15.2 gene serves as the only detection index or one of the effective detection indexes.

The present invention also provides the following method:

The present invention provides a method for treating or assisting in treating tumors, comprising the following steps: using the above inhibitor alone to treat the tumor.

The present invention provides a method for treating or assisting in treating tumors, comprising the following steps: using the above inhibitor in combination with other tumor treatment drugs to treat tumors.

The present invention provides a method for treating or assisting in treating tumors, comprising the following steps: using the above inhibitor in combination with other tumor treatment methods to treat tumors.

The present invention provides a method for assisting in evaluating the prognosis of a tumor patient after chemotherapy treatment, comprising the following steps: detecting the expression level of CTD-2256P15.2 in the tumor tissue of the tumor patient;

The prognosis of patients with high expression of CTD-2256P15.2 is worse than or potentially worse than that of patients with low expression of CTD-2256P15.2.

The present invention provides a method for evaluating or assisting in evaluating the sensitivity of a tumor patient to a chemotherapy drug, comprising the following steps: detecting the expression level of CTD-2256P15.2 in the tumor tissue of the tumor patient;

Patients with high expression levels of CTD-2256P15.2 are less sensitive to chemotherapy drugs or less selective than patients with low expression levels of CTD-2256P15.2.

The above-mentioned substance for detecting the expression level of CTD-2256P15.2 in tumor tissue is specifically the primer used for fluorescent quantitative PCR in the example.

The prognostic status is specifically reflected in the disease-free survival and/or overall survival, specifically:

The overall survival of tumor patients corresponding to tumor tissues with high expression of CTD-2256P15.2 gene after chemotherapy is shorter or potentially shorter than that of tumor patients corresponding to tumor tissues with low expression of CTD-2256P15.2 gene;

Or, the disease progression-free survival period after chemotherapy of a tumor patient corresponding to a tumor tissue with a high expression level of the CTD-2256P15.2 gene is shorter or potentially shorter than that of a tumor patient corresponding to a tumor tissue with a low expression level of the CTD-2256P15.2 gene.

The above-mentioned chemotherapy drugs are tumor treatment drugs that can cause DNA damage, including but not limited to anthracyclines, camptothecins or PARP inhibitors;

Furthermore, drug resistance is resistance to chemotherapeutic drugs.

CTD-2256P15.2 is highly expressed in a variety of tumor types and can be induced to express by a variety of DNA-damaging chemotherapy drugs. CTD-2256P15.2 can encode a functional micropeptide PACMP. On the one hand, PACMP competitively inhibits the binding and ubiquitination degradation of CtIP, an important factor in the homologous recombination repair pathway, by binding to the substrate adaptor protein KLHL15 of the ubiquitin ligase Cul3. On the other hand, it promotes PAR signal amplification by binding to the PAR chain induced by chemotherapy and DNA damage. The two synergistically promote the growth and drug resistance of tumor cells. Therefore, CTD-2256P15.2 or its encoded micropeptide PACMP is a new target for inhibiting tumor growth and enhancing the clinical treatment effect of tumors.

The inhibitor of CTD-2256P15.2 or its encoded micropeptide PACMP provided by the present invention can significantly inhibit the growth of tumor cells, increase the apoptosis of tumor cells, reduce the volume of tumors, and have excellent anti-tumor effects when acting on tumor cells or tumor tissues. The novel anti-tumor drug combination provided by the present invention combines CTD-2256P15.2 or its encoded micropeptide PACMP inhibitor with other anti-tumor drugs, which can significantly enhance the killing effect of anti-tumor drugs on tumor cells and reduce the chemotherapy resistance of tumor cells, thereby improving the clinical treatment effect of tumors. CTD-2256P15.2 is highly expressed in chemotherapy-resistant tumor tissues and cell lines, and its high expression is significantly negatively correlated with the disease-free survival and overall survival of tumor patients. The CTD2256P15.2 gene expression level provided by the present invention can be used as a molecular indicator for predicting the sensitivity and prognosis of tumor patients to chemotherapy, which creates a new standard for effectively guiding the clinical chemotherapy medication of tumor patients and evaluating the prognosis of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that CTD-2256P15.2 is highly expressed in the epirubicin-resistant breast cancer cell line MCF7-EPI.

FIG. 2 shows that the expression of CTD-2256P15.2 is negatively correlated with the prognosis of breast cancer patients.

Figure 3 shows that knocking down CTD-2256P15.2 can enhance the sensitivity of various tumor cells to epirubicin.

Figure 4 shows that knocking down CTD-2256P15.2 can increase the sensitivity of tumor cells to multiple treatment options (including chemotherapy, targeted therapy and radiotherapy).

Figure 5 shows that CTD-2256P15.2 can encode micropeptides.

FIG. 6 shows that CTD-2256P15.2 regulates the chemotherapy sensitivity of tumor cells through its encoded micropeptide.

FIG. 7 shows that inhibiting CTD-2256P15.2 or its encoded micropeptide can significantly inhibit tumor growth and enhance the sensitivity of chemotherapeutic drugs.

Figure 8 shows that inhibiting CTD-2256P15.2 or its encoded micropeptide can reduce the efficiency of homologous recombination repair and microhomologous end joining repair, reduce CtIP protein levels and DNA damage-induced PAR levels.

DETAILED DESCRIPTION

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

The overall survival (OS) in the following examples is defined as the time from enrollment to death due to any cause or the last follow-up.

The overall survival rate in the following embodiments is defined as the probability that a patient will survive to a certain time after being followed up from a certain time point.

The progression-free survival (PFS) in the following examples is defined as the period from the start of treatment to the observation of disease progression or death due to any cause in patients with tumor diseases.

The disease-free survival rate in the following examples is defined as the probability that no disease progression is observed at a certain time after the patient is followed up from a certain time point.

Example 1. Long noncoding RNA CTD-2256P15.2 and its use as a marker for predicting chemotherapy prognosis

1. Discovery of long noncoding RNA CTD-2256P15.2

By performing transcriptome sequencing on breast cancer tumor tissues that are sensitive and resistant to the anthracycline chemotherapy drug epirubicin, we identified that the long noncoding RNA CTD-2256P15.2 is highly expressed in epirubicin-resistant tumor tissues.

The nucleotide sequence of the long non-coding RNA CTD-2256P15.2 is sequence 1 in the sequence list.

2. Verification of the relationship between CTD-2256P15.2 expression and tumor drug resistance

The CCK8 method was used to detect the drug resistance of tumor cells to epirubicin, as follows:

MCF7-EPI cells in the logarithmic phase (cells resistant to epirubicin obtained by gradually increasing the concentration of epirubicin (from 20nM to 500nM) during the culture of MCF7 cells) and control cells MCF7 (ATCC Cat#HTB-22, RRID:CVCL_0031) were inoculated into 96-well plates, 4000 cells per well. After 12 hours of culture, different concentrations of epirubicin (EPI) were added to the culture system of each culture well. After continuing to culture for 24 hours, the culture medium was aspirated, and fresh culture medium containing 10% (volume percentage) CCK8 reagent (DOJINDO, Cat#CK04) was added and incubated for 4 hours. Then the absorbance value of each well at 465nm was detected with an ELISA instrument to draw the relative survival curve of the cells.

The results are shown in FIG1A , and it can be seen that as the concentration of EPI increases, the survival rate of the epirubicin-resistant cell line MCF7-EPI is higher than that of the cell line MCF7, which proves that MCF7-EPI is resistant to EPI.

Total RNA from MCF7-EPI cells and MCF7 cell lines was extracted using Trizol reagent, and the extracted RNA was reverse transcribed into cDNA using reverse transcriptase. Then, the RNA level of CTD-2256P15.2 was detected using real-time fluorescence quantitative PCR.

The primers for real-time fluorescence quantitative PCR detection were CTD-2256P15.2: forward primer: GACTTCTGCATTTGGCTGGAAGG, reverse primer: CTAACTCAGGGTATCGGAACCGA; internal reference GAPDH: forward primer: GGAGCGAGATCCCTCCAAAAT, reverse primer: GGCTGTTGTCATACTTCTCATGG.

The results are shown in Figure 1B, and it can be seen that the expression of CTD-2256P15.2 in MCF7-EPI was significantly higher than that in the control MCF7 cells.

3. Relationship between the expression of CTD-2256P15.2 and chemotherapy sensitivity in tumor patients

In order to further verify the relationship between the expression of CTD-2256P15.2 and the chemotherapy sensitivity of tumor patients, tumor tissues of 92 breast cancer patients who received epirubicin-based chemotherapy regimens (from Tianjin Medical University Cancer Hospital) were collected. Among them, 48 patients had no tumor progression after chemotherapy and had a good prognosis, which was the good prognosis group; 44 patients had disease progression after chemotherapy and had a poor prognosis, which was the poor prognosis group (Tables 1 and 2).

Table 1 shows the expression results of tumor tissues of 44 breast cancer patients with poor prognosis

In the above table, the recurrence status of 1 in the third column indicates recurrence during the follow-up period in the second column, and 0 indicates no recurrence or loss to follow-up during the follow-up period in the second column; the death status of 1 in the fifth column indicates death during the follow-up period in the fourth column, and 0 indicates no death or loss to follow-up during the follow-up period in the fourth column.

Table 2 shows the expression results of tumor tissues of 48 breast cancer patients with good prognosis

In the above table, the recurrence status of 1 in the third column indicates recurrence during the follow-up period in the second column, and 0 indicates no recurrence or loss to follow-up during the follow-up period in the second column; the death status of 1 in the fifth column indicates death during the follow-up period in the fourth column, and 0 indicates no death or loss to follow-up during the follow-up period in the fourth column.

The above 92 breast cancer tumor tissue samples were formalin-fixed paraffin-embedded tissue sections. The tumor tissue RNA was extracted using the NucleoSpin total RNA extraction kit. The specific extraction steps were referred to the instructions for use of the kit. The extracted RNA was then reverse transcribed into cDNA using reverse transcriptase, and then the expression of CTD-2256P15.2 in these tumor tissues was detected using real-time fluorescence quantitative PCR.

The method of fluorescence quantitative PCR technology is the same as the above two real-time fluorescence quantitative PCRs.

Patients whose expression levels were higher than the median expression level of all patients (median value was 1.15) were classified as the high-expression patient group, and patients whose expression levels were lower than or equal to the median expression level of all patients were classified as the low-expression patient group.

The results are shown in Figure 2, where A is the expression level of CTD-2256P15.2 in patients with poor prognosis and good prognosis, B is the progression-free survival (PFS), and C is the overall survival (OS). It can be seen that the expression of CTD-2256P15.2 in the tumor tissues of patients with poor prognosis is significantly higher than that in the tissues of patients with good prognosis (Figure 2A); by analyzing the prognosis of these tumor patients, it was found that compared with patients with low expression of CTD-2256P15.2, patients with high expression of CTD-2256P15.2 in tumor tissues had shorter progression-free survival (PFS, Figure 2B, reflected in progression-free survival rate) and overall survival (OS, Figure 2C, reflected in overall survival rate), and a poorer prognosis, reflecting that patients with high expression of CTD-2256P15.2 have poor drug sensitivity, leading to disease progression.

Therefore, detecting the expression of CTD-2256P15.2 gene in tumor tissue can predict the sensitivity of tumor patients to chemotherapy drugs; or detecting the expression of CTD-2256P15.2 gene in tumor tissue can predict the prognosis of tumor patients, which is specifically reflected in the disease-free survival and/or overall survival;

Tumor patients whose tumor tissues have high expression of CTD-2256P15.2 gene have lower sensitivity to chemotherapy drugs than tumor patients whose tumor tissues have low expression of CTD-2256P15.2 gene;

The prognosis of tumor patients whose tumor tissues have high expression of CTD-2256P15.2 gene after chemotherapy is worse than that of tumor patients whose tumor tissues have low expression of CTD-2256P15.2 gene;

Specifically, the overall survival of tumor patients whose tumor tissues have high expression of the CTD-2256P15.2 gene after chemotherapy is shorter than that of tumor patients whose tumor tissues have low expression of the CTD-2256P15.2 gene;

Or, the disease progression-free survival period after chemotherapy of a tumor patient corresponding to a tumor tissue with a high expression level of the CTD-2256P15.2 gene is shorter than that of a tumor patient corresponding to a tumor tissue with a low expression level of the CTD-2256P15.2 gene.

Example 2: CTD-2256P15.2 as a target for tumor treatment

1. Expression of CTD-2256P15.2 in various tumors

In order to verify that inhibiting CTD-2256P15.2 can increase the sensitivity of various tumors to chemotherapeutic drugs, the expression of CTD-2256P15.2 in various tumors was first analyzed.

The transcriptome sequencing quantitative data of various tumors in TCGA were retrieved on the GePIA website. The Ensembl ID of CTD-2256P15.2 gene is ENSG00000259802. By searching this ID, the expression of CTD-2256P15.2 in various tumor types can be obtained. BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; CESC: cervical squamous cell carcinoma and adenocarcinoma; COAD: colon cancer; DLBC: diffuse large B-cell lymphoma; ESCA: esophageal cancer; GBM: multiforme glioma; HNSC: head and neck squamous cell carcinoma; LGG: low-grade glioma of the brain; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic cancer; PRAD: prostate cancer; READ: rectal adenocarcinoma; STAD: gastric cancer; THYM: thymic carcinoma; UCEC: endometrial carcinoma; UCS: uterine sarcoma.

The results are shown in Figure 3A, TPM: Transcripts Per Million; it can be seen that the expression of CTD-2256P15.2 gene in tumor tissues of multiple tumor types such as breast cancer, lung cancer, gastric cancer, and ovarian cancer is higher than that in normal tissues.

Therefore, CTD-2256P15.2 can be selected as a therapeutic target in multiple tumors for the following experiments.

2. Application of CTD-2256P15.2 inhibitor siRNA in improving the sensitivity of tumor cells to chemotherapy

1. Preparation of CTD-2256P15.2 inhibitor siRNA

In order to detect the regulatory effect of CTD-2256P15.2 on the chemotherapy sensitivity of tumor cells, two specific siRNAs targeting CTD-2256P15.2 (CTD-2256P15.2 inhibitor siRNAs) silnc15.2-1 and silnc15.2-2 were designed and synthesized according to the RNA sequence of CTD-2256P15.2. The sequences are:

silnc15.2-2,GCGGCUUCUGGAGGGACAA (sequence 2);

silnc15.2-1,GCAGAUGACCUAGCACAAA (sequence 3);

The sequence of the control siRNA (siNC) is: UUCUCCGAACGUGUCACGU.

2. Transfection of siRNA and chemotherapy sensitivity experiment

Lipofectmine RNAiMAX (Invitrogen, Cat#13778150) was used to transfect control siRNA (siNC), specific silnc15.2-1 and silnc15.2-2 targeting CTD-2256P15.2 into breast cancer cells (MCF7ATCC Cat#HTB-22, RRID:CVCL_0031, MDA-MB-231ATCC Cat#CRM-HTB-26, RRID:CVCL_0062), osteosarcoma cells (U2OS Cat#HTB-96; RRID:CVCL_0042), gastric cancer (AGS ATCC Cat#CRL-1739, RRID:CVCL_0139), cervical cancer cells (HeLa Cat#CRL-7923; RRID:CVCL_0030), ovarian cancer (SK-OV-3Cat#HTB-77; RRID:CVCL_0532) and non-small cell lung cancer cells (A549 Cat#CCL-185; RRID:CVCL_0023), the siRNA working concentration was 100nM; specifically, a 3ml transfection system was used in a 6cm culture dish (about 10^6 cells), in which the siRNA working concentration was 100nM for knockdown.

After 48 hours of transfection with different siRNAs, some cells were trypsinized and collected by centrifugation. Total cellular RNA was extracted and reverse transcribed into cDNA. Fluorescence quantitative PCR was used to detect the level of CTD-2256P15.2 in transfected cells.

Fluorescence quantitative PCR detection method Example 1, the results of transfecting MCF7 cells with different siRNAs are shown in Figure 3B, and it can be seen that silnc15.2-1 and silnc15.2-2 achieve knockdown of CTD-2256P15.2 in tumor cells and reduce its expression level.

At the same time, 24 hours after transfection, some cells were inoculated into 96-well plates. After the cells adhered to the wall, different concentrations of epirubicin were added to the cell culture system for continuous treatment for 24 hours, and the survival of the cells was detected by CCK8 method (the method was the same as that of Example 1).

The CCK8 method was used to detect the survival of different tumor cells transfected with CTD-2256P15.2 inhibitor siRNA. The results are shown in Figures 3C-3I. It can be seen that compared with the control siRNA, the knockdown of CTD-2256P15.2 in different tumor cells by CTD-2256P15.2 inhibitory siRNA can significantly enhance the sensitivity of these cells to epirubicin and reduce the survival rate of tumor cells, indicating that CTD-2256P15.2 is an effective target for enhancing the killing effect of tumor chemotherapy drugs and improving clinical treatment effects.

Therefore, siRNA knocking out or inhibiting the expression of CTD-2256P15.2 can enhance the sensitivity of tumor cells to chemotherapeutic drugs and reduce the survival rate of tumor cells after treatment with chemotherapeutic drugs.

III. Combination of CTD-2256P15.2 inhibitory siRNA and other treatments

According to the above two methods, the synthetic control siRNA (siNC), specific silnc15.2-1 and silnc15.2-2 targeting CTD-2256P15.2 were transfected into MCF7 cells using lipofectmine RNAiMAX (Invitrogen, Cat#13778150) to make the siRNA working concentration be 100 nM.

24 hours after transfection, the transfected cells were inoculated into a 96-well plate. After the cells adhered to the wall, different concentrations of camptothecin (CPT) (Sigma, Cat#C9911), ATR inhibitor (VE-822) (Selleck, Cat#S7102) or CDK4/6 inhibitor (Palbociclib, Selleck, Cat#S1116) were added to the culture system of some cells. After continuous treatment for 48 hours, the cell survival was detected using the CCK8 reagent method, and the detection method was the same as in Example 1.

The results are shown in Figures 4A-4C, where A, B, and C are siRNA+camptothecin (CPT), siRNA+ATR inhibitor (VE-822), and siRNA+CDK4/6 inhibitor (Palbociclib), respectively. It can be seen that after CTD-2256P15.2 inhibitory siRNA inhibits the expression of CTD-2256P15.2, it can significantly increase the sensitivity of MCF7 cells to tumor therapeutic drugs such as camptothecin (CPT), ATR inhibitor (VE-822) and CDK4/6 inhibitor (Palbociclib).

Therefore, these results show that the combination of CTD-2256P15.2 inhibitory siRNA and other chemotherapeutic regimens or chemotherapy drugs can significantly enhance the killing effect on tumor cells, providing a new strategy for improving the therapeutic effect of tumors. In other words, CTD-2256P15.2 inhibitory siRNA can enhance the killing effect of anti-tumor drugs on tumor cells and reduce the chemotherapy resistance of tumor cells.

IV. Application of CTD-2256P15.2 inhibitor shRNA in improving the sensitivity of tumor cells to chemotherapy

The expression level of CTD-2256P15.2 was reduced by shRNA technology, and the survival of tumor cells after Olaparib or X-ray treatment was detected by cloning experiments. The specific steps are as follows:

1. Design and synthesize two shRNA coding sequences for targeting CTD-2256P15.2, including shlnc15.2-2: GCGGCTTCTGGAGGGACAAAGCTCGAGCTTTGTCCCTCCAGAAGCCGC (sequence 4); shlnc15.2-1: GCAGATGACCTAGCACAAATACTCGAGTATTTGTGCTAGGTCATCTGC (sequence 5).

shNC sequence: TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA

They were respectively connected to the pLKO vector (Addgene Cat#10878 https://www.addgene.org/protocols/plko/) to obtain the recombinant pLKO plasmid for expressing shRNA.

The recombinant pLKO-shlnc15.2-2 plasmid is a vector obtained by replacing the coding gene of shlnc15.2-2 shown in sequence 4 between AgeI and EcoRI sites of the pLKO vector. This vector expresses shlnc15.2-2 (RNA encoded by sequence 4).

The recombinant pLKO-shlnc15.2-1 plasmid is a vector in which the coding gene of shlnc15.2-1 shown in SEQ ID NO: 5 is replaced between AgeI and EcoRI sites of the pLKO vector. The vector expresses shlnc15.2-1 (RNA encoded by SEQ ID NO: 5).

The recombinant pLKO-shNC plasmid is a vector obtained by replacing the shNC coding gene between the AgeI and EcoRI sites of the pLKO vector.

2. Lentivirus packaging and infection

One day before transfection, 293T cells (Cat#CRL-3216; RRID:CVCL_0063) in the logarithmic growth phase were seeded in a 10 cm culture dish to a cell density of about 50%, and plasmid transfection was performed after culturing overnight.

Before transfection, the culture medium was replaced with fresh culture medium without antibiotics and placed at 37 degrees. According to the instructions of the vigoFect transfection reagent, 5 μg of plasmid (the above-mentioned pLKO-shlnc15.2-1 or pLKO-shlnc15.2-2 and the viral packaging plasmid pRSV-Rev (Addgene ID 12253), pMD2.G (Addgene ID 12259), pMDLg/pRRE (Addgene ID 12251) (ratio of 2:1:1:1) was transferred into 293T cells. After 24 hours of transfection, the culture medium was discarded and 8 ml of fresh complete culture medium was added to continue culturing. After 48 hours, the cell culture supernatant contained the corresponding lentivirus.

One day in advance, MCF7 cells were inoculated into 6 cm culture dishes to a density of 50% for infection with lentivirus. When infected on the second day, the 293T culture supernatant was collected and filtered through a 0.45 μM filter into a clean centrifuge tube. The filtered virus was mixed with an equal volume of fresh culture medium, and polybrene was added at a final concentration of 10 μg/mL. After mixing, it was added to MCF7 cells and the medium was changed 8 hours after infection. After continuing to culture for 48 hours, 1 mg/ml puromycin was added for screening. When the cells can grow stably in puromycin and qPCR can detect a good knockdown effect, it is considered that MCF7 cells that stably express shRNA (shCTD-2256P15.2)-1/2 targeting CTD-2256P15.2 are obtained, and they are named MCF7 shRNA-1 and MCF7shRNA-2.

The cells transformed with the recombinant pLKO-shNC plasmid were used as a control and named MCF7 shNC.

The above-mentioned qPCR detection method is as follows: extract the total RNA of the above-mentioned cells, use reverse transcriptase to reverse transcribe the RNA into cDNA, and use fluorescent quantitative PCR to detect the content of CTD-2256P15.2 (primers are the same as before). The results are shown in Figure 4D. It can be seen that shlnc15.2-1 and shlnc15.2-2 can knock out CTD-2256P15.2 and reduce its expression.

3. Function

MCF7 shRNA-1 or MCF7 shRNA-2 obtained in 2 above and MCF7 shNC (control cells) were seeded in 6 cm culture dishes, respectively.

After the cells adhered, some of about 500 cells were treated with different concentrations of the PARP inhibitor Olaparib for 24 hours, the culture medium was discarded, the cells were washed twice with PBS, fresh complete culture medium was added, and the cells were placed in a 37-degree incubator for further culture for 14 days. The culture medium was discarded, the cells were gently rinsed with water twice, the culture dish was dried at room temperature, the number of cell clones visible to the naked eye was counted, and the clone survival ratio was calculated according to the formula (survival ratio = the number of clones formed under different treatment conditions divided by the number of clones formed under the same group without treatment conditions), and the clone survival curve was drawn.

Alternatively, about 500 adherent cells were irradiated with different doses of X-rays to cause DNA damage, and then cultured for 14 days, the number of cell clones was counted, and a clone survival curve was drawn.

The results are shown in Figures 4E and 4F. Using shRNA to reduce the expression of CTD-2256P15.2 in MCF7 cells can significantly enhance the killing effect of PARP inhibitors and X-rays on cells, reduce tumor cell survival, and increase the sensitivity of cells to PARP inhibitor chemotherapy and radiation therapy.

The above results show that the combination of CTD-2256P15.2 inhibitor shRNA and other chemotherapeutic regimens or chemotherapy drugs can significantly enhance the killing effect on tumor cells, providing a new strategy for improving the effect of tumor treatment. In other words, CTD-2256P15.2 inhibitor shRNA can enhance the killing effect of anti-tumor drugs or tumor treatment methods on tumor cells and reduce the chemotherapy resistance of tumor cells.

Example 3: Micropeptide encoded by CTD-2256P15.2 and its regulation of chemotherapy sensitivity of tumor cells

1. Micropeptide encoded by CTD-2256P15.2

Through bioinformatics prediction, it was found that the open reading frame 1 (ORF1, i.e., positions 138-272 of sequence 1) of CTD-2256P15.2 may encode a micropeptide with a length of 44 amino acids, and the amino acid sequence of the micropeptide is MAASGGTKKAQSGGRRLREPSSRPSRRARQRPRRGALRKAGRFL (sequence 8).

In order to verify the coding nature of CTD-2256P15.2, a sequence containing the SBP-FLAG tag was knocked into the 3' end (before the stop codon) of the ORF1 of the CTD-2256P15.2 gene in U2OS cells using CRISPR-Cas9 technology. Monoclonal cells with successful insertion were obtained by picking monoclonal cells and performing PCR sequencing.

The specific method is as follows:

1) Design and synthesize a DNA sequence that specifically targets the 3’ end of ORF1: CGGCGTGCACTGTCGGTCGGCGG (sequence 6), clone this DNA into the pX330 vector, and use it to transcribe and generate gRNA.

The recombinant vector pX330-gRNA is a vector obtained by inserting the DNA molecule represented by CGGCGTGCACTGTCGGTCGGCGG (SEQ ID 6) into the Bbs1 site of the pX330 vector (Addgene ID 42230), which expresses gRNA (gRNA encoded by SEQ ID 6).

2) Design and synthesize donor DNA:

The donor DNA consists of left homology arm-tag-containing sequence (SBP-FLAG-P2A-puromycin)-right homology arm, and the nucleotide sequence is sequence 7.

3) Type in

The donor DNA and pX330-gRNA plasmid were electroporated into U2OS cells (ATCC, Cat#HTB-96; RRID: CVCL_0042) using a Pulse Generator-CUY21EDDID 2 electroporation instrument at a voltage of 135 V. After 48 hours, 1 mg/ml puromycin was added for screening, and then the cells grown after puromycin screening were inoculated for monoclonal inoculation. After 2 weeks, monoclonal cells were picked and expanded, and then genomic DNA was extracted, and PCR amplification and sequencing were used to identify the positive clone U2OS-KI cells with successful knock-in.

The primers used in the above PCR amplification are: forward primer: AGAGGCTGACAGAAAGCGAG, reverse primer: CTAACTCAGGGTATCGGAACCGA, and a fragment of approximately 1580 bp was obtained and sequenced. The positive clones with successful knock-in were named U2OS-KI cells, which express the fusion protein ORF1-SBP-FLAG-P2A (the encoding nucleic acid of the fusion protein is sequence 10, which is the sequence obtained by fusing the 3' end (before the stop codon) of the first open reading frame of CTD-2256P15.2 to SBP-FLAG-P2A-puromycin. During protein translation, the P2A sequence undergoes self-cleavage to obtain the ORF1-SBP-FLAG-P2A fusion protein).

4) shCTD-2256P15.2 cells

The shRNA technology was used to knock down CTD-2256P15.2 in the U2OS-KI cells obtained in 3) above. The method was referred to Example 2 (IV). The cells obtained after U2OS-KI cells were transfected with the recombinant pLKO-shlnc15.2-1 plasmid by lentivirus were named U2OS-KI-shlnc15.2.

The cells obtained by transfecting U2OS-KI cells with the recombinant pLKO-shNC plasmid via lentivirus were named U2OS-KI-shNC.

5) Detection of expression

A. Verify the endogenous expression of ORF1 in U2OS-KI control cells. The specific method is as follows:

(1) Immunofluorescence: U2OS cells (control cells), U2OS-KI-shlnc15.2 and U2OS-KI-shNC cells in the logarithmic phase were seeded on glass slides at a density of 60-80%. On the second day, the culture medium was aspirated, the cells were washed twice with PBS, and 4% paraformaldehyde solution was added to fix the cells at room temperature for 20 minutes. The fixative was discarded and the cells were washed three times with PBS. A PBS solution containing 0.5% Triton X-100 was added to treat the cells at room temperature for 10 minutes, the Triton solution was discarded, the cells were washed three times with PBS, and a PBS solution containing 5% BSA was added to block the cells at room temperature for 1 hour. The cells were then incubated with the primary anti-SBP tag antibody (Santa Cruz, Cat#sc-101595; RRID:AB_1128239) and the fluorescent secondary antibody (Life Technologies, Cat#A11029; RRID:AB_138404) in sequence, and finally the slides were sealed with a blocking agent containing DAPI.

The expression of ORF1 fusion protein was detected under a microscope.

The results are shown in Figure 5A. WT is U2OS cells, KI-shlnc15.2 is U2OS-KI-shlnc15.2 cells, and KI-shNC is U2OS-KI-shNC cells. It can be seen that in U2OS-KI-shNC cells, ORF1 can initiate translation and express ORF1-SBP-FLAG-P2A fusion protein, while there is no expression of the fusion protein in the lnc15.2 knockdown cells U2OS-KI-shlnc15.2.

(2) Immunoprecipitation: 4×10 ⁶ logarithmic growth phase U2OS, U2OS-KI-shNC, and U2OS-KI-shlnc15.2 cells were collected, lysed with lysis buffer containing 1% Triton and centrifuged, anti-FLAG beads were added to the obtained cell lysate, and incubated at 4 degrees for 2 hours. The supernatant was discarded after centrifugation, and the beads were washed with lysis buffer, and 2×SDS sample buffer was added and boiled at 95 degrees for 10 minutes. The expression of ORF1 fusion protein was detected by western blot technology.

The results are shown in Figure 5B. In U2OS-KI-shNC, ORF1 was able to initiate translation and express ORF1-SBP-FLAG-P2A fusion protein. The expression of ORF1-SBP-FLAG-P2A in sh15.2 knockdown cells U2OS-KI-shlnc15.2 was significantly reduced compared with that in U2OS-KI-shNC cells.

These results indicate that ORF1 of CTD-2256P15.2 can encode a micropeptide of 44 amino acids in length.

2. CTD-2256P15.2 regulates the chemotherapy sensitivity of tumor cells through its encoded micropeptide

In order to verify the regulatory effect of the micropeptide encoded by CTD-2256P15.2 on the chemotherapy sensitivity of tumor cells, the wild-type full-length (FL) and full-length (FL*) of CTD-2256P15.2 with ORF1 start codon mutation that are resistant to shCTD-2256P15.2 were complemented in MCF7 and U2OS cells with stable knockdown of CTD-2256P15.2, and the ORF1 fusion protein was complemented to detect the sensitivity of cells to epirubicin.

The specific method is as follows:

1. Construction of MCF7 and U2OS cells with stable knockdown of CTD-2256P15.2

MCF7 and U2OS cells with stable knockdown of CTD-2256P15.2 were constructed using shRNA technology, and the specific steps were referred to Example 2. The difference was that the cells used were MCF7 and U2OS cells, and the recombinant plasmid was pLKO-shlnc15.2-1 plasmid, and MCF7 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2 and U2OS cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2 were obtained.

The recombinant pLKO-shNC plasmid knockout was used as a control to obtain control shNC-expressing MCF7 cells and control shNC-expressing U2OS cells.

2. Construction of complementing cell lines

The specific steps are as follows:

1) The shRNA-resistant CTD-2256P15.2 wild-type full-length (FL), the full-length (FL*) with a mutation in the start codon of ORF1 (ATG mutated to ATT, which can no longer start encoding micropeptides), and the ORF1 fusion protein (ORF1 C-terminal fusion SBP-FLAG tag) were connected to the pNL lentiviral expression vector (pNL-EGFP/CMV/WPREdU3, Addgene, #17579), and the recombinant plasmids were screened as follows:

The recombinant plasmid FL is a vector obtained by cloning the gene sequence of CTD-2256P15.2 (sequence 1) into the Nhe1 and EcoR1 restriction sites of the pNL lentiviral expression vector;

The recombinant plasmid FL* is a vector obtained by cloning the ORF1 start codon mutation sequence of CTD-2256P15.2 (the sequence is ATG of the sequence shown at positions 138-141 of SEQ ID NO. 1 into ATT, i.e., the start codon ATG of the open reading frame 1 into ATT) into the Nhe1 and EcoR1 restriction sites of the pNL lentiviral expression vector;

The recombinant plasmid ORF1 is a vector obtained by cloning the coding gene of the ORF1-SBP-FLAG fusion protein (whose sequence is sequence 9, i.e., positions 138-269 of sequence 1 + SBP-FLAG nucleic acid sequence) into the Nhe1 and EcoR1 restriction sites of the pNL lentiviral expression vector;

2) The above-mentioned recombinant plasmids were co-transfected with the packaging plasmid into 293T cells by the lentiviral packaging and infection method (the method is the same as that of Example 2, step 2) to obtain a culture supernatant containing virus particles; the lentivirus was then used to infect MCF7 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2 and U2OS cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2 to obtain stable complementation cells, which were named MCF7 cells (transfected with recombinant plasmid ORF1 complementation shRNA (shCTD-2256P15.2)-1) respectively. RF1), U2OS cells with ORF1 complemented shRNA (shCTD-2256P15.2)-1 (transferred into recombinant plasmid ORF1), MCF7 cells with Fl* complemented shRNA (shCTD-2256P15.2)-1 (transferred into recombinant plasmid FL*), U2OS cells with Fl* complemented shRNA (shCTD-2256P15.2)-1 (transferred into recombinant plasmid FL*), MCF7 cells with Fl complemented shRNA (shCTD-2256P15.2)-1 (transferred into recombinant plasmid FL) and U2OS cells with Fl complemented shRNA (shCTD-2256P15.2)-1 (transferred into recombinant plasmid FL).

The above cells were seeded into a 96-well plate, and the attached cells were treated with 1 μM epirubicin for 24 hours, and the cell survival rate was detected using CCK8 reagent, using the same method as in Example 1. MCF7 cells expressing shNC control, U2OS cells expressing shNC control, MCF7 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2, and U2OS cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2 were used as controls.

The results are shown in Figure 6, A is MCF7 cells, B is U2OS cells; shNC represents cells expressing shNC, shlnc15.2 represents cells stably expressing shRNA targeting CTD-2256P15.2 (shCTD-2256P15.2)-1, shlnc15.2+ORF1 represents cells complemented with ORF1 (shCTD-2256P15.2)-1, shlnc15.2+FL represents cells complemented with FL ( The cells expressed as shCTD-2256P15.2)-1, shlnc15.2+FL* indicated that FL* complemented shRNA(shCTD-2256P15.2)-1. It can be seen that knocking down CTD-2256P15.2 can increase the sensitivity of tumor cells to doxorubicin, complementing the expression of wild-type full-length (ORF1) and ORF1 fusion protein (FL) can restore the reduced sensitivity of cells to doxorubicin, however, complementing the expression of the full-length ORF1 mutation (FL*) cannot restore it.

The above results indicate that CTD-2256P15.2 regulates the chemotherapy sensitivity of tumor cells through the micropeptide it encodes.

3. Verification of the regulatory effect of CTD-2256P15.2 or its encoded micropeptide on tumor chemotherapy sensitivity at the animal level

The triple-negative breast cancer cell line MDA-MB-231 with stable knockdown of CTD-2256P15.2 and full-length wild-type or full-length mutant ORF1 was constructed, and a certain amount of cells were inoculated under the mammary fat pad of female nude mice. When the transplanted tumor grew to a certain size, the mice were treated with normal saline and epirubicin, and the size of the tumor was measured and counted. At the end of the experiment, the mice were killed and the tumors were dissected out, photographed, and the tumor weight was measured.

The details are as follows:

1) MDA-MB-231 cells with stable knockdown of CTD-2256P15.2 were constructed using shRNA, and MDA-MB-231 cells with wild-type full-length (FL) complementation or full-length mutant (FL*) complementation of ORF1 start codon mutation (from ATG to ATT) were constructed in knockdown cells by lentiviral infection, as follows:

Referring to Example 2, the recombinant pLKO-shlnc15.2-1 plasmid was transferred into the MDA-MB-231 cell line to obtain MDA-MB-231 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2;

The recombinant pLKO-shNC plasmid was transfected as a control to obtain control knockdown MDA-MB-231 cells expressing shNC.

Referring to 2 of the above, the wild-type lnc15.2 full-length (FL) and mutant lnc15.2 full-length (FL*) were respectively transferred into MDA-MB-231 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2 to obtain MDA-MB-231 cells with FL complemented shRNA (shCTD-2256P15.2)-1 and MDA-MB-231 cells with FL* complemented shRNA (shCTD-2256P15.2)-1.

2) 4×10 ⁶ of the above cells in the logarithmic growth phase were mixed with an equal volume of matrix gel and inoculated under the mammary fat pad of female BALB/c nude mice. When the long diameter of the tumor reached 5 mm, the mice were randomly divided into two groups and injected intraperitoneally with 5 mg/kg of epirubicin or an equal volume of normal saline. The drug was administered once every 4-5 days, the tumor volume was measured every other day, and the relative growth curve of the tumor was drawn. The mice were killed on the 11th day after the first administration, the tumor was dissected, photographed and the tumor weight was measured. MDA-MB-231 cells expressing shNC and MDA-MB-231 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2 were used as controls.

The results are shown in Figure 7, A-C are respectively (A) the growth of tumor tissue of each group of transplanted tumor cells under the condition of treatment or no treatment with epirubicin, (B) the weight of each group of transplanted tumors collected, and (C) the photos of each group of transplanted tumors collected. shNC indicates MDA-MB-231 cells expressing shNC, shlnc15.2 indicates MDA-MB-231 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2, shlnc15.2+FL indicates complementation of wild-type full-length lnc15.2 in shlnc5.2 MDA-MB-231 cells, and shlnc15.2+FL* indicates complementation of mutant full-length lnc15.2 in shlnc5.2 MDA-MB-231 cells. Whether from the tumor volume, tumor or tumor size, it can be seen that knocking down CTD-2256P15.2 significantly inhibits tumor growth and increases sensitivity to epirubicin, and complementation of wild-type rather than mutant full-length can restore tumor growth and sensitivity to epirubicin.

This suggests that inhibiting CTD-2256P15.2 or its encoded micropeptide has excellent clinical application potential. Inhibition alone can reduce tumor growth, while combined use with other chemotherapeutic drugs can enhance the killing effect of chemotherapeutic drugs and improve clinical treatment effects.

4. The mechanism of action of CTD-2256P15.2 or its encoded micropeptide in regulating tumor growth and sensitivity to chemotherapeutic drugs was studied

1) DNA homologous recombination repair (HR) efficiency and microhomology-mediated end joining (MMEJ) detection

In DR-U2OS cells (described in the following literature: Xia, B., Sheng, Q., Nakanishi, K., Ohashi, A., Wu, J., Christ, N., Liu, X., Jasin, M., Couch, F. J., and Livingston, D. M. (2006). Control of BRCA2 cellular and clinical functions by a nuclear partner, PALB2. Mol Cell 22, 719-729.) and U2OS EGFP-MMEJ cells (described in the following literature: Wang, H., Shao, Z., Shi, L. Z., Hwang, P. Y., Truong, L. N., Berns, M. W., Chen, D. J. C., and Wu, X. (2012). CtIP protein dimerization is critical for its recruitment to chromosomal DNA double-stranded breaks. J Biol Chem. 287 (25), 21471-21480.), silnc15.2-1, silnc15.2-2 and siRNA (siNC) were transfected into cells respectively (the method was the same as that of Example 2, to obtain silnc15.2-1 knockdown DR-U2OS cells, silnc15.2-2 knockdown DR-U2OS cells, siNC knockout DR-U2OS cells, silnc15.2-1 knockdown MMEJ cells, silnc15.2-2 knockdown MMEJ cells and siNC knockout MMEJ cells.

The introduced siBRCA1 (UAUAAGACCUCUGGCAUGAAU) and siCtIP (GCUAAAACAGGAACGAAUC) were used as positive controls.

GFP-positive cells were sorted by flow cytometry, and GFP-positive cells and total cells were counted respectively. The ratio of positive cells to total cells was the efficiency of DNA homologous recombination repair (HR) or microhomology-mediated end joining (MMEJ) efficiency.

The results are shown in Figures 8A (DR-U2OS cells) and 8B (U2OS EGFP-MMEJ cells). After knocking down CTD-2256P15.2 using siRNA, the efficiency of DNA homologous recombination repair (HR) and microhomology-mediated end joining (MMEJ) was significantly reduced.

2) Protein content detection of CtIP, a key factor in HR repair

The above-mentioned two prepared MCF7 cells stably expressing shRNA (shCTD-2256P15.2)-1 targeting CTD-2256P15.2, MCF7 cells with ORF1 complement shRNA (shCTD-2256P15.2)-1 (transferred into recombinant plasmid ORF1), and MCF7 cells with Fl* complement shRNA (shCTD-2256P15.2)-1 (transferred into recombinant plasmid FL*) were collected in the logarithmic growth phase, and the protein level of intracellular CtIP was detected by western blot.

The results are shown in Figure 8C. After using shRNA to inhibit the expression of CTD-2256P15.2, the protein content of CtIP, a key factor in HR repair, in MCF7 cells was significantly reduced. The protein level of CtIP can be restored by complementing the expression of the wild-type full-length instead of the ORF1 mutant full-length.

3) PAR detection

The pNL lentiviral empty expression vector (i.e., the pNL lentiviral expression vector), recombinant plasmid FL and recombinant plasmid FL* were respectively transferred into the silnc15.2-1 knockdown MCF7 cells to obtain MCF7 cells with pNL lentiviral empty expression vector complemented with silnc15.2-1, MCF7 cells with FL complemented with silnc15.2-1 and MCF7 cells with FL* complemented with silnc15.2-1.

The cells in the logarithmic growth phase were inoculated into a 6-well plate to a cell density of about 70%. On the second day, the culture medium was discarded, and the cells were treated with serum-free medium containing 400 μM H ₂ O ₂ at 37 degrees for 5 minutes. The culture medium was discarded, the cells were washed twice with PBS, and 1× SDS sample buffer was added to lyse the cells. The level of intracellular PAR was detected by western blot. Serum-free medium without H ₂ O ₂ was used as a control.

The results are shown in Figure 8D, siNC is MCF7 cells treated with siNC, silnc15.2 is MCF7 cells treated with silnc15.2-1, - is MCF7 cells complemented with silnc15.2-1 by the pNL lentiviral empty expression vector, FL is MCF7 cells complemented with silnc15.2-1 by FL, and FL* is MCF7 cells complemented with silnc15.2-1 by FL*; it can be seen that knocking down CTD-2256P15.2 can significantly inhibit the generation of DNA damage-induced poly ADP ribose (PAR) modified chains, the wild-type full-length can restore PAR generation, and the ORF1 mutant full-length cannot restore PAR (Figure 8D).

The above results indicate that CtIP is a key factor in the normal initiation of DNA homologous recombination repair and microhomology-mediated end-joining repair functions in cells, and the two DNA damage repair pathways are impaired after CtIP loss. The simultaneous downregulation of CtIP and/or PAR caused by inhibiting CTD-2256P15.2 or its encoded micropeptide PACMP can achieve the effect of inhibiting tumor growth through the effect of synthetic lethality.

SEQUENCE LISTING

<110>Institute of Zoology, Chinese Academy of Sciences; Beijing Institute of Genomics, Chinese Academy of Sciences (National Center for Bioinformatics); Tianjin Cancer Hospital (Tianjin Medical University Cancer Hospital)

<120> Application of CTD-2256P15.2 and its encoded micropeptide as targets in the development of tumor therapeutic drugs

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<170> PatentIn version 3.5

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acaatgcgag tctgcgcccc acgctcccgc accgtcaatg cgagtctgcg ccccacgctc 1320

ccgcaccgtg atgccgaggc caaatggcta gagtgggccc aggcatcagc atttctgttt 1380

ggcagccggg cagttgtggt gcacgttttg aggaactact aaagcctgcc tgtgatgaaa 1440

aaggcaaaag ctgacttcac caaaattact cccagggaga cttctgcatt tggctggaag 1500

gacatttgag taaaccgctg aggctggtgg ttgaaacata atcctctaag ggagatcggt 1560

tccgataccc tgagttag 1578

<210> 8

<211> 44

<212> PRT

<213> Artificial sequence

<400> 8

Met Ala Ala Ser Gly Gly Thr Lys Lys Ala Gln Ser Gly Gly Arg Arg

1 5 10 15

Leu Arg Glu Pro Ser Ser Arg Pro Ser Arg Arg Ala Arg Gln Arg Pro

20 25 30

Arg Arg Gly Ala Leu Arg Lys Ala Gly Arg Phe Leu

35 40

<210> 9

<211> 270

<212> DNA

<213> Artificial sequence

<400> 9

atggcggctt ctggagggac aaagaaggcg cagagcggtg ggaggaggct gcgagaacct 60

agctcccgcc ccagccgacg tgcgagacaa aggccccggc gcggggctct tcggaaggcc 120

gggaggttcc tggacgagaa gaccaccggc tggcggggcg gccacgtggt ggagggcctg 180

gccggcgagc tggagcagct gcgggccagg ctggagcacc accctcaggg ccagcggggag 240

cccgattaca aggatgacga cgataagtaa 270

<210> 10

<211> 339

<212> DNA

<213> Artificial sequence

<400> 10

atggcggctt ctggagggac aaagaaggcg cagagcggtg ggaggaggct gcgagaacct 60

agctcccgcc ccagccgacg tgcgagacaa aggccccggc gcggggctct tcggaaggcc 120

gggaggttcc tgggcagcgg catggacgag aagaccaccg gctggcgggg cggccacgtg 180

gtggagggcc tggccggcga gctggagcag ctgcgggcca ggctggagca ccaccctcag 240

ggccagcggg aggattacaa ggatgacgac gataagggaa gcggagctac taacttcagc 300

ctgctgaagc aggctggaga cgtggaggag aaccctgga 339

Claims (3)

1. Use of an inhibitor having any of the following functions a-b in at least one of the following 1)-6): 1) Preparation of tumor treatment products; 2) Prepare products that reduce the resistance of tumor cells to DNA-damaging chemotherapy drugs; 3) Prepare products that increase the sensitivity of tumor cells to DNA-damaging chemotherapy drugs; 4) Preparation of products that inhibit tumor resistance caused by the DNA homologous recombination repair pathway; 5) Prepare products that inhibit tumor resistance caused by the microhomology-mediated end-joining repair pathway in tumor cells; 6) Preparation of products that inhibit tumor resistance caused by DNA damage-induced poly (ADP-ribose) chain generation; a. Inhibit the expression of CTD-2256P15.2 gene; b. Inhibit the biological function of the micropeptide PACMP encoded by the CTD-2256P15.2 gene; The inhibitor is siRNA or shRNA; The nucleotide sequence of the CTD-2256P15.2 gene is sequence 1 or positions 138-272 of sequence 1 in the sequence list; The tumor is invasive breast cancer, gastric cancer, osteosarcoma, cervical squamous cell carcinoma and adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma or ovarian serous cystadenocarcinoma. 2. Use of the inhibitor of claim 1, and other tumor therapeutic drugs or reagents or instruments required for other anti-tumor therapeutic methods in at least one of the following: 1) Preparation of tumor treatment products; 2) Prepare products that reduce the resistance of tumor cells to DNA-damaging chemotherapy drugs; 3) Prepare products that increase the sensitivity of tumor cells to DNA-damaging chemotherapy drugs; The other tumor therapeutic drugs are camptothecin, PARP inhibitor, ATR inhibitor or CDK4/6 inhibitor; The reagent or instrument required for the other anti-tumor treatment method is X-ray; The tumor is invasive breast cancer, gastric cancer, osteosarcoma, cervical squamous cell carcinoma and adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma or ovarian serous cystadenocarcinoma. 3. A product comprising the inhibitor of claim 1, and other tumor therapeutic drugs or reagents or instruments required for other anti-tumor therapeutic methods; The product has at least one of the following functions: 1) Cancer treatment; 2) Reduce the resistance of tumor cells to DNA-damaging chemotherapy drugs; 3) Increase the sensitivity of tumor cells to DNA-damaging chemotherapy drugs; The other tumor therapeutic drugs are camptothecin, PARP inhibitor, ATR inhibitor or CDK4/6 inhibitor; The reagent or instrument required for the other anti-tumor treatment method is X-ray; The tumor is invasive breast cancer, gastric cancer, osteosarcoma, cervical squamous cell carcinoma and adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma or ovarian serous cystadenocarcinoma.

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